Nitrogen assimilation is of fundamental importance to the growth of plants. Of all the mineral nutrients required by plants, nitrogen is required in the greatest abundance. The main forms of nitrogen taken up by plants in the field are nitrate and ammonia, the principle components of nitrogenous fertilizers. Plants take up either nitrate or ammonium ions from the soil, depending on availability. Nitrate will be more abundant in well-oxygenated, non-acidic soils, whilst ammonium will predominate in acidic or water-logged soils. Experiments on growth parameters of tobacco clearly demonstrated that relative growth rate, chlorophyll content, leaf area and root area increased dramatically in response to increasing nitrate supply.
Plants have developed a very efficient nitrogen uptake system in order to cope with the large variation in nitrate content of cultivated soils. Plant roots take up nitrate and ammonia by the action of specific nitrate transporters (NTR). Two nitrate transport systems have been shown to coexist in plants, the NRT1 gene family and the NRT2 gene family. It is believed that both transporter families act cooperatively to take up nitrate from soil and distribute it around plants. It is generally assumed that the NRT1 gene family mediates the root low-affinity transport system (LATS, i.e. when external nitrate concentration >1 mM), while the NRT2 gene family mediates the high affinity transport system (HATS, i.e. when external nitrate concentration <1 mM). In Arabidopsis, seven genes are known to belong to the NRT2 gene family. It is also known that expression of these genes is regulated by the concentration of nitrate to which a plant is exposed.
However, excluding Atnrt2.1, Atnrt2.4 and Atnrt2.7, the function of the other NRT2 genes has not been elucidated.
One of the best characterized members of the NRT2 gene family is Atnrt2.1. AtNRT2.1 interacts with NAR2 (AtNRT3) protein to form a major component of the HATS in Arabidopsis. Over-expression of a mutated, non-functional form of the AtNRT2.1 nitrate transporter protein in Arabidopsis causes a significant reduction in the activity of the high-affinity nitrate-uptake system and a reduction in leaf nitrate content. As a consequence, the growth of these Nrt2.1 mutant plants was severely impaired when Arabidopsis was grown in low nitrate concentrations.
Nrt2.7 is a poorly characterized gene. It is known to be expressed in plant seeds, specifically in the vacuolar membrane, and it regulates the nitrate content of plant seeds. The nitrate transporter Nrt2.4 has been recently characterized. The NRT2.4 protein is involved in the uptake of nitrate by the roots of plants, at very low external concentrations, and in the loading of nitrates into the phloem of plant shoots.
The regulation of the activities of nitrate transporters, and nitrate and nitrite reductases is critical in controlling primary nitrogen assimilation throughout the plant, and has a significant impact on the growth and development of the plant. However, under certain conditions, nitrate may accumulate, mainly in green photosynthetically active tissues, where it is stored in the vacuoles of the mesophyll cells. High levels of nitrate accumulation can occur during periods of low temperature and/or solar irradiation (for example, in greenhouse crops during the winter), when there is less photosynthetic capacity to assimilate the stored nitrate, or as a result of high nitrate levels in the soil.
An increase in nitrate levels can have a number of deleterious consequences, not only in terms of plant growth, but also in terms of human or animal health where the plant is consumed, as well as environmental consequences. Many of the adverse consequences of nitrate accumulation are mediated through the production of nitrite.
Therefore, to prevent nitrate accumulation, one strategy would be increasing nitrogen remobilisation in plants, for example when they become senescent, which could have important applications in crop production. Firstly, nitrogen remobilised from leaves can be transported to the younger leaves as well as the developing seed. Increasing the efficiency of nitrogen exit from senescent leaves could therefore potentially increase nitrogen supply to seeds and younger parts of the plant, and thereby increase crop yield and nitrogen use efficiency. This is clearly a valuable goal when the world population is increasing but crop yields are not increasing sufficiently to meet demand. One potential target crop is Brassica napus (oilseed rape), which has poor nitrogen efficiency due to poor nitrogen remobilisation from vegetative tissue. Another target crop is wheat, as the potential benefits of increasing grain protein content are great. Grain protein content not only affects nutritive value of wheat, but also determines grain usage and therefore market value. For example, increased grain protein content results in increased bread volume.
Also, an ability to increase nitrogen remobilisation could be very useful in the tobacco industry because it is known that residual nitrogen in tobacco leaves contributes to the formation of nitrosamines, as illustrated in FIG. 1. In particular, nitrate and nitrite act as precursors to tobacco-specific nitrosamine (TSNA) formation in cured leaf. The processing of the tobacco leaves by the tobacco industry involves the removal of petioles and midribs of the cured leaves which are believed to act as nitrate storage organs, devoid of flavour and high in TSNAs.
Also, the formation of nitrosamines in the stomach is a result of endogenous nitrosation. Oral bacteria chemically reduce nitrate consumed in food and drink to nitrite, which can form nitrosating agents in the acidic environment of the stomach. These react with amines to produce nitrosamines and cause DNA strand breaks or cross linking of DNA. Another problem associated with an excess of nitrate is the formation of methaemoglobin which gives rise to blue baby syndrome, where the oxygen carrying capacity of haemoglobin is blocked by nitrite, causing chemical asphyxiation in infants.
As a consequence of these health concerns, a number of regulatory authorities have set limits on the amount of nitrate allowed in leafy green vegetables such as spinach and lettuce (e.g. European Commission Regulation 653/2003), depending on the time of harvest. These limits have resulted in any produce with a high nitrate content being unmarketable. Consequently, there have been efforts to reduce nitrate content of plants by managing the application of nitrogen-containing fertilisers or improved systems of crop husbandry. Some authorities have also set limits on the amounts of nitrate in drinking water.
There is therefore a need for means for alleviating the adverse effects associated with nitrate accumulation in plants. With this in mind, the inventors have developed a genetic construct, which may be used in the preparation of transgenic plants, which exhibits surprisingly reduced nitrate concentrations.